Increasingly, oxidative stress has been implicated in a variety of disease states, leading widespread interest in determining relevant biomarkers for evaluation of oxidative stress. Glutathione (GSH) is a tripeptide of glutamic acid, cysteine, and glycine, with a gamma-glutamyl linkage between Cys and Glu and the free Cys sulfhydryl as the functionally active component. GSH acts as an antioxidant to protect cells or tissues from oxidation by reactive oxygen/nitrogen species. Upon exposure to oxidative conditions, GSH is oxidized to form glutathione disulfide (GSSG), which can be subsequently converted back to GSH by glutathione reductase. An increased GSH-to-GSSH ratio has been used as a sensitive biomarker to evaluate extent of oxidative stress [1, 2]. Most commercially available GSH/GSSG assay kits are based on enzymatic recycling methods [3]. The variation of the quantification is high due to high susceptibility of GSH to artificial oxidation during the performance of the assay. For example, one group reported that the mean values obtained for GSH and GSSG among thirty studies spanned two orders of magnitude [3]. Furthermore, GSH is mostly utilized as an intracellular antioxidant, so the ratio of GSH/GSSH can be used to assess oxidative state in whole blood or red blood cell samples. However, the substantially lower extracellular concentration of GSH/GSSG makes it a much less sensitive indicator for other types of samples such as plasma. Protein bound GSH has long been considered an indicator of oxidative stress in whole blood [4] [5], but much less attention is paid to protein bound Cysteine. The reason, at least, in part, is the lack of a sensitive and high throughput assay to determine it.
Thus, more reliable and sensitive assays for determining the level of oxidation of thiol and disulfide containing molecules in a sample are needed that can be used for a variety of patient samples, including plasma. Furthermore, finding plasma biomarkers of thiol oxidation are needed for evaluation of a wide range of disease status with oxidative stress and to evaluate response to antioxidants treatment. The present disclosure satisfies these and other needs.